Recombinant porcine adenovirus vector

ABSTRACT

This invention relates to a recombinant vector including a recombinant porcine adenovirus, stably incorporating and capable of expression of at least one heterologous nucleotide sequence. The nucleotide sequence is preferably one which encodes an antigenic determinant of Hog Cholera Virus or Pseudorabies virus. The further invention relates to a method of production of recombinant vectors, to methods of preparation of vaccines based on the vectors, to administration strategies and to methods of protecting pigs from disease.

FIELD OF INVENTION

This invention relates to delivery vectors for antigen producing genes(heterologous gene sequences or fragments thereof) used to generateimmune responses in commercial pigs susceptible to decimation bydisease. Such vectors are especially useful for the preparation ofvaccines which can be easily administered on a large scale to protectpigs against disease. This invention also relates to a method ofproduction of suitable delivery vectors, to methods of preparation ofvaccines based on the vectors, to administration strategies and to amethod protecting pigs from disease.

BACKGROUND

The productivity of the intensive pig industry depends on the control ofinfectious diseases. Whilst diseases can be controlled in part by goodhygiene and quarantine measures, the industry must still rely onvaccination to protect herds. In a commercial situation, the cost peranimal is high in terms of feed and current disease control costs andtherefore, the costs in disease prevention and control by any newlyproposed vaccine must be cheap, effective and easy to deliver.

Conventionally, vaccines constituting live viral particles have beenprepared by virus passage and selection of attenuated forms.Alternatively, killed vaccines were prepared from virulent viruses.

The most recent description of the use of viral vectors in the controlof disease in pigs was the deletion mutant of pseudorabies virus for thecontrol of Aujesky's disease. The use of a herpesvirus as a vector hasthe advantage of being able to stimulate a humoral and cell-mediatedresponse, thus providing possible life long protection. Anotheradvantage is the ability to insert other heterologous sequences in thisvector, being expressed from a suitable promoter, to produce antigensfor exposure to the animals immune system, thus protecting against twodiseases. There are disadvantages of this system. Firstly, there is theissue of latency. Herpesviruses have the ability to intergrate into theneurons in ganglia for the life of the animal. It only requires asuitable stress on the animal to cause the reactivation of the virus andconsequently full disease. However, it is now known that the deletion ofa specific gene, glycoprotein E, will attenuate the virus and preventreactivation from latency. Therefore, this deletion vector is now widelyused as an eradication vector for Aujesky's disease and subsequentlywill not be available as a suitable vector for the delivery of otherantigens.

It is thus the aim of this invention to provide a delivery vehicle forheterologous sequences of genetic material that is particularly suitedto administration on a large scale.

In particular, it is the aim of this invention to provide or enhancemeans for generation and/or optimisation of antibodies or cell-mediatedimmunity so as to provide protection against infection with commonporcine diseases. It is an additional aim to provide a process forpreparation of a suitable means for generation and/or optimisation ofantibodies or cell-mediated immunity so as to protect pigs againstinfection with common porcine diseases. It is a further aim to provide aprotection strategy.

SUMMARY OF INVENTION

The invention provides, in one embodiment, a recombinant porcineadenovirus capable of expressing DNA of interest said DNA of interestbeing stably integrated into an appropriate site of said recombinantporcine adenovirus genome.

In another embodiment the invention provides a recombinant vectorincluding a recombinant porcine adenovirus which stably incorporates atleast one heterologous nucleotide sequence. Preferably the heterologousnucleotide sequence is capable of expression as an antigenicpolypeptide. The antigenic polypeptide encoded by at least onenucleotide sequence is preferably foreign to the host vector.

In a further embodiment of the present invention the heterologousnucleotide sequence is capable of expression as an immuno-potentiatormolecule.

It is also to be understood that the heterologous nucleotide sequencemay encode for and/or express, an antigenic polypeptide and animmuno-potentiator molecule.

The recombinant vector may comprise a live recombinant porcineadenovirus in which the virion structural proteins are unchanged fromthose in the native porcine adenovirus from which the recombinantporcine adenovirus is produced.

This invention is partially predicated on the discovery that there arenon-essential regions in the porcine adenovirus genome which do notcorrespond to those characterised previously on other adenoviruses thusmaking this virus particularly suited to delivery of heterologoussequences.

This invention is also predicated on the discovery that the porcineadenovirus generates a prolonged response in pigs thus making it wellsuited as a vaccine vehicle. Furthermore, the existence of a number ofserotypes specific to respiratory or gastrointestinal tracts, allows theselection of a vaccine vehicle suited to a target organ and the type ofimmune response required.

The invention is also predicated on the discovery that porcineadenovirus can package genomic DNA greater than the 105% rule formammalian adenoviruses with intermediate size genomes and that theresultant packaged virions are stable in vitro and in vivo.

Adenoviruses are a large and diverse family, having been isolated frommany living species, including man and other mammals as well as avariety of birds. As a result adenoviruses have been separated into atleast two genera, the Mastoadenoviridae and the Aviadenoviridae, andmore recently a third genera has been proposed, the Atadenovirdae, whichincludes some bovine and avian adenoviruses (egg drop syndrome) (Benköand Harrach, Archives of Virology 143, 829-837, 1998).

Porcine adenoviruses are prevalent infectious agents of pigs and to datefour distinct serotypes have been recognised (Adair and McFerran, 1976)and evidence for at least one more (Derbyshire et al, 1975). Of the fourserotypes found, three (serotypes 1 to 3) were isolated from thegastrointestinal tract while the fourth was recovered from therespiratory system. The porcine adenoviruses are considered to be a lowpathogenic widespread agent and although isolations were made in generalfrom diseased animals, it was most likely that the adenovirus waspresent only as a secondary infection. They have been isolated from pigswith diarrhoea and respiratory infections but it has been consideredthat at least the gastrointestinal adenovirus infections are usuallyasymptomatic (Sanford and Hoover, 1983). Porcine adenoviruses are spreadby ingestion or inhalation and experimental infection via oral,intranasal and intratracheal inoculations have resulted in uptake of thevirus. Experimental pathogenicity studies have shown that the primarysites of infection are the lower small intestine probably the tonsil(Sharpe and Jessett, 1967; Shadduck et al., 1968). With serotype 4infection, a viraemia appears to develop in experimental infections.However, this may be a less common manifestation with thegastrointestinal serotypes (Shadduck et al, 1968). Faecal excretion isthe most common cause for spread of PAV, being present for several weekspost infection. Nasal shedding also occurs under experimentalconditions. PAV's role in pneumonia has been suggested to be that ofeither a predisposing factor or a synergist (Kasza et al., 1969;Schiefer et al., 1974) but experimental pneumonia with serotype 4 didnot require a second agent to produce disease (Smith et al., 1973).

Porcine adenoviruses have yet to be examined in much detail and littleis known about their role in disease or how common they are. This is dueto the fact that they do not produce any significant disease in herdsand have failed to draw the interest of industry through loss ofproduction. It is likely that the number of serotypes of porcineadenoviruses is much greater than four and that it probably exists inthe majority of pig herds as a normal commensal.

Work done on porcine adenovirus in regards to its morphology andmolecular biology, has shown some similarities with otherMastadenoviruses examined. Its morphology is that of other adenovirusesexamined with an icosahedral capsid containing a core of a doublestranded DNA genome. Very little work on the characterisation of the PAVgenome has been published (Benkö et al, 1990, Kleiboeker et al., 1993,Reddy et al, 1993, Kleiboeker, 1994). The size of the PAV genome(approx. 34.8 kb) is slightly smaller than that of human adenoviruses(approx. 35.9 kb). One study has shown using hybridisation with DNAprobes from the total genome of human adenovirus type 2 that there isreasonable DNA homology between the porcine and human adenoviruses(Benkö et al., 1990). A recent report on the serotype 4 PAV demonstratedthat its genomic layout was also similar to that of the humanadenoviruses in the area of the L4 and E3 regions (including the 33K andpVIII genes) even though the sequence homology was not as strong as mayhave been expected (Kleiboeker, 1994).

While choosing appropriate PAV for development as a live vectors todeliver vaccines to pigs, it is important to take into account thenatural prevalence of serotypes. Those serotypes not commonlyencountered in the field have an obvious advantages over those to whichpigs are frequently exposed and to which they may have developedimmunity.

A further consideration is the ability of the vector to remain active inthe pig beyond the period which maternal antibodies in colostrum protectpigs immediately post-birth.

Other important considerations in choosing potential PAV vectors arepathogenicity and immunogenicity. Preferably live vector viruses shouldbe highly infectious but non-pathogenic (or at least attenuated) suchthat they do not themselves adversely affect the target species.

The preferred candidates for vaccine vectors are non-pathogenic isolatesof serotype 4 (respiratory) and serotype 3 (gastrointestinal). Serotype3 has been chosen as the serotype of choice due to excellent growthabilities in continuous pig kidney cell lines. The isolation of otherserotypes, which seems likely, may well alter this selection. It isnotable that the more virulent strains produce a greater antibodyresponse.

Heterologous nucleotide sequences which may be incorporated intonon-essential regions of the viral genome and which may encode theantigenic determinants of infectious organisms against which thegeneration of antibodies or cell-mediated immunity is desirable may bethose expressing antigenic determinants of intestinal infections causedby gastrointestinal viruses; for example rotavirus or parvovirusinfections, or respiratory viruses, for example parainfluenza virus, orthat of Japanese encephalitis.

Heterologous nucleotide sequences which may be incorporated include theantigenic determinants of the agents of:

-   -   Porcine parvovirus    -   Mycoplasma hyopneumonia    -   Porcine parainfluenza    -   Transmissable gastroenteritis (porcine coronavirus)    -   Porcine rotavirus    -   Hog cholera virus (Classical swine fever)    -   Swine dysentery    -   African swine fever virus    -   Pseudorabies virus (Aujesky's disease virus) in particular, the        glycoprotein D of the pseudorabies virus    -   Porcine respiratory and reproductive syndrome virus (PRRSV)    -   Heterologous nucleotide sequences more preferred for        incorporation in the vectors of the invention are those        expressing antigenic determinants of porcine parvovirus, porcine        rotavirus, porcine coronavirus and classical swine fever virus.

It is also envisaged the heterologous sequences incorporated may beimmuno-potentiator molecules such as cytokines or growth promoters, forexample porcine interleukin 4 (IL4), gamma interferon (γIFN),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), FLT-3 ligand and interleukin 3(IL-3).

The type of immune response stimulated by candidate vectors may affectthe selection of heterologous nucleotide sequences for insertiontherein. PAV serotypes 1, 2 and 3, which naturally infect via the gutmay induce local mucosal immunity and are thus more suitable forinfections of the intestines (eg classical swine fever virus). PAVserotype 4, which naturally infects via the respiratory system, may bemore suitable for infections of the respiratory tract (eg porcineparainfluenza) may also induce good local immunity.

The DNA of interest which may comprise heterologous genes coding forantigenic determinants or immuno-potentiator molecules may be located inat least one non-essential region of the viral genome.

Non-essential regions of the viral genome which may be suitable for thepurposes of replacement with or insertion of heterologous nucleotidesequences may be non-coding regions at the right terminal end of thegenome at map units 97 to 99.5. Preferred non-coding regions include theearly region (E3) of the PAV genome at map units 81-84.

The heterologous gene sequences may be associated with a promoter andleader sequence in order that the nucleotide sequence may be expressedin situ as efficiently as possible. Preferably the heterologous genesequence is associated with the porcine adenoviral major late promoterand splice leader sequence. The mammalian adenovirus moor late promoterlies near 16-17 map units on the adenovirus genetic map and contains aclassical TATA sequence motif (Johnson, D. C., Ghosh-Chondhury, G.,Smiley, J. R., Fallis, L. and Graham, F. L. (1988), Abundant expressionof herpes simplex virus glycoprotein gB using an adenovirus vector.Virology 164, 1-14).

The splice leader sequence of the porcine adenovirus senotype underconsideration is a tripartite sequence spliced to the 5′ end of the mRNAof all late genes.

The heterologous gene sequence may also be associated with a polyadenylation sequence.

Instead of the porcine adenoviral major late promoter, any othersuitable eukaryotic promoter may be used. For example, those of SV40virus, cytomegalovirus (CMV) or human adenovirus may be used.

Processing and poly adenylation signals other than those of porcineadenoviruses may also be considered, for example, that of SV40.

In a further aspect of the invention there is provided a recombinantvaccine for generating and/or optimising antibodies or cell-mediatedimmunity so as to provide or enhance protection against infection withan infectious organism in pigs, the vaccine including at least onerecombinant porcine adenovirus vector stably incorporating at least oneheterologous nucleotide sequence formulated with suitable carriers andexcipients. Preferably the nucleotide sequence is capable of expressionas an antigenic polypeptide or as an immuno-potentiator molecule. Morepreferably, the heterologous nucleotide sequence may encode for and/orexpress, an antigenic polypeptide and an immuno-potentiator molecule.

The antigenic polypeptide encoded by the at least one nucleotidesequence is preferably foreign to the host vector. At least onenucleotide sequence may be associated with a promoter/leader and a polyA sequence.

The recombinant vaccine may include live recombinant porcine adenovirusvector in which the virion structural proteins are unchanged from thatin the native porcine adenovirus from which the recombinant porcineadenovirus is produced.

Preferred vector candidates for use in the recombinant vaccine are PAVisolates of serotype 3 and 4. Use of other serotypes is possible,depending on herd existing immunity and its environment

The vaccine may be directed against respiratory and intestinalinfections caused by a variety of agents. In order to direct the vaccineagainst a specific infectious organism, heterologous gene sequencesencoding the antigenic determinants of those infectious organisms may beincorporated into non-essential regions of the genome of the porcineadenovirus comprising the vector. If the vaccine is to be used tooptimise protection against disease, suitable heterologous nucleotidesequences may be those of immuno-potentiators such as cytokines orgrowth promoters.

The vaccine may comprise other constituents, such as stabilisers,excipients, other pharmaceutically acceptable compounds or any otherantigen or part thereof. The vaccine may be in the form of a lyophilisedpreparation or as a suspension, all of which are common in the field ofvaccine production.

A suitable carrier for such as a vaccine may be isotonic bufferedsaline.

In a further aspect of the invention, there is provided a method ofpreparing a vaccine for generation and/or optimisation of antibodies orcell-mediated immunity so as to induce or enhance protection against aninfectious organism in a pig, which includes constructing a recombinantporcine adenovirus vector stably incorporating at least one heterologousnucleotide sequence, and placing said recombinant porcine adenovirusvector in a form suitable for administration. Preferably the nucleotidesequence is capable of expression as an antigenic polypeptide althoughit may also be an immuno-potentiator molecule. More preferably, thenucleotide sequence may encode for and/or express, an antigenicpolypeptide and an immuno-potentiatormolecule. The nucleotide sequenceis conveniently foreign to the host vector.

Even more preferably, the nucleotide sequence is associated withpromoter/leader and poly A sequences.

The form of administration may be that of an enteric coated dosage unit,an inoculum for intra-peritoneal, intramuscular or subcutaneousadministration, an aerosol spray, by oral or intranasal application.Administration in the drinking water or in feed pellets is alsopossible.

In another aspect of the invention, there is provided a method ofproducing a porcine adenovirus vaccine vector which includes insertinginto a porcine adenovirus at least one heterologous nucleotide sequence.Said heterologous nucleotide sequence is preferably capable ofexpression as an antigenic polypeptide although it may also be animmuno-potentiator molecule. More preferably, the nucleotide sequencemay encode for and/or express, an antigenic polypeptide and animmuno-potentiator molecule.

Preferably the antigenic polypeptide encoded by the at least onenucleotide sequence is foreign to the host vector.

More preferably, the heterologous nucleotide sequence is associated withpromoter/leader and poly A sequences.

In one method of construction of a suitable vector the non-essentialregion to be altered to incorporate foreign DNA could be constructed viahomologous recombination. By this method the non-essential region iscloned and foreign DNA together with promoter, leader and polyadenylation sequences is inserted preferably by homologous recombinationbetween flanking sequences. By this method also, deletion of portions ofthe non-essential region is possible to create extra room for larger DNAinserts that are beyond the normal packing constraints of the virus.

By this method a DNA expression cassette containing an appropriate PAVpromoter with foreign gene sequence as well as leader sequences and polyadenylaton recognition sequences can be constructed with the uniquerestriction enzyme sites flanking the cassette enabling easy insertioninto the PAV genome.

In another aspect of the invention there is provided strategies foradministration of the vaccines of the invention.

In one strategy, a heterologous antigen and immuno-modulatory moleculesuch as a cytokine may be expressed in the same recombinant anddelivered as a single vaccine.

In one strategy according to the invention PAV vector based vaccines maybe administered as ‘cocktails’ comprising 2 or more virus vectorscarrying different foreign genes or immuno-potentiators.

In a preferred vaccination strategy of the invention, the ‘cocktail’ orsimultaneous strategy, a vaccine based on both PAV serotype 3 andserotype 4 is used.

In another preferred strategy, a base recombinant serotype 3 porcineadenovirus is constructed and the fiber gene from serotype 4 replacingthat of serotype 3 or the fiber from serotype 4 additionally cloned intothe vaccine to broaden the targeting of the invention to both gut andrespiratory delivery.

In an alternative strategy according to the invention, PAV vector basedvaccines may be administered consecutively of each other to eitheradminister booster vaccines or new vaccines at some stage subsequent toinitial PAV vaccination, The vaccines used are preferably based onheterologous PAV isolates.

In a preferred version of the “consecutive” strategy, vaccines based onisolates serotypically unrelated are selected so as to achieve maximumprotection against infection. In one example of such a strategy avaccine based on PAV serotype 3 is administered subsequently or prior tovaccination with a vaccine based on PAV serotype 4.

Pigs are conveniently inoculated with vector vaccines according to theinvention at any age. Piglets may be vaccinated at 1 day old, breedersmay be vaccinated regularly up to point of giving birth and thereafter.

Preferably according to either the consecutive strategy or the cocktailstrategy, pigs are vaccinated while still not fully immunocompetent.More conveniently, day-old pigs can be vaccinated for protection againstre-infection after a period of 4 weeks subsequent to initialvaccination.

In a further embodiment of the invention there is provided a method forproducing an immune response in a pig including administering to the pigan effective amount of a recombinant vaccine according to the invention.An effective amount is an amount sufficient to elicit an immuneresponse, preferably at least 10⁴ TCID₅₀ per dose.

The vaccine of the invention may of course be combined with vaccinesagainst other viruses or organisms such as parvovirus or Aujesky'sdisease at the time of its administration.

In a preferred aspect of this embodiment of the invention,administration is by oral delivery or intra-nasally.

Methods for construction and testing of recombinant vectors and vaccinesaccording to this invention will be well known to those skilled in theart. Standard procedures for endonuclease digestion, ligation andelectrophoresis were carried out in accordance with the manufacturer'sor suppliers instructions. Standard techniques are not described indetail and will be well understood by persons skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the DNA restriction endonuclease map of the entirePAV serotype 3 genome.

FIG. 2 illustrates the sequence characterisation and cloning of themajor later promoter and splice leader sequences of PAV serotype 3.

FIG. 3 illustrates the sequences of the major later promoter, upstreamenhancer sequence and splice leaders 1, 2 and 3.

FIG. 4 illustrates the terminal 720 bases of the right end of thegenome.

FIG. 5 illustrates the promoter region of E3 and the overlapping L4area.

FIG. 6 illustrates a preferred method of construction of a PAV vector.

FIG. 7 represents temperature data of pigs vaccinated with a PAV basedvaccine following challenge with CSFV antigen.

FIG. 8 graphically represents anti-PAV antibody levels detected by ELISAin pigs pre and post vaccination with a PAV based vaccine.

FIG. 9 graphically illustrates the development of neutralisingantibodies in pigs vaccinated with a PAV based vaccine pre and postchallenge with CSFV antigen.

FIG. 10 graphically illustrates the mean white blood cell (WBC) countsof pigs vaccinated with a recombinant PAV vaccine expressing porcineG-CSF.

FIG. 11 graphically illustrates the percentage change in white bloodcell (WBC) counts following vaccination with a recombinant PAV vaccineexpressing porcine G-CSF.

FIG. 12 graphically represents the percentage change in monocyte cellpopulations following vaccination with recombinant PAV-G-CSF.

FIG. 13 graphically represents the percentage change in lymphocyte cellpopulations following vaccination with recombinant PAV-G-CSF.

FIG. 14 graphically represents the change in stimulation of T-cellsfollowing vaccination with recombinant PAV-G-CSF.

FIGS. 15 a, b and c graphically illustrate a method of construction of aPAV E3 vector.

PREFERRED EMBODIMENTS

Aspects of preferred embodiments of the invention based on PAV isolatesserotype 3 and serotype 4 will now be described. Whilst these twoisolates have been selected because of their sites of infection in thepig, it will be appreciated that other isolates of porcine adenovirusmay be more suitable for construction of vaccine vectors provided thecriteria for selection described herein before are met.

In general, PAV are considered of low pathogenicity with littleconsequence in the field. The pathogenic significance of PAV is reviewedin Derbyshire, 1989. The first report of isolation of PAV was from a 12day old pig with diarrhoea (Haig et al., 1964). Two years later, PAVtype 4 was first reported, isolated from the brain of a pig sufferingfrom encephalitis of unknown cause (Kasza, 1966). Later reports haveassociated PAV mainly with diarrhoea in the field although this isnormally low grade. PAV can also be regularly isolated from healthyanimals with no disease signs and it is quite likely that its isolationfrom diseased animals is more a coincidence of its prevalence than anindicator of pathogenicity. However, an association between serotype 4and respiratory disease has been reported (Watt, 1978) and this has beensupported by experimental infection (Edington et al, 1972). Experimentalinfections with gastrointestinal serotypes of the virus (eg serotype 3)have been able to produce diarrhoea but the pathological changesproduced were not clinically significant

The genome of the selected PAV serotype 3 was characterised byconventional methods. The DNA restriction endonuclease maps of theentire genome is illustrated in FIG. 1. The genomes are orientated leftto right. By convention adenovirus genomes are normally orientated suchthat the terminal region from which no late mRNA transcripts aresynthesised is located at the left end. The enzymes used to generate themap are indicated at the edge of each map.

Characterisation of Major Late Promoter (MLP) and Splice LeaderSequences (LS) of PAV Serotype 3

Identification and cloning of the PAV MLP

By use of restriction enzyme and genetic maps of the PAV serotype 3genome, a region was located that contained the MLP and leader sequences(FIG. 1). The fragments identified in this region were cloned intoplasmid vectors and sequenced.

The MLP promoter sequence was identified as containing a classical TATAsequence, the only one in the region sequenced, as well as upstreamfactors and was subsequently confirmed by the location of the leadersequence and the transcriptional start site.

FIGS. 2 and 3 illustrate the sequence characterisation of the major latepromoter and splice leader sequences of PAV serotype 3.

In order to determine the structure and sequence of the leader sequencespliced to late mRNA, porcine kidney cells were infected with PAV andthe infection was allowed to proceed until late in the infection cycle(usually 20-24 hr p.i.). At this time total RNA was purified from theinfected cells using the RNAgents total RNA purification kit (Promega).The isolated RNA was precipitated with isopropanol and stored at −70° C.in 200 μl aliquots until required. Poly A (mRNA) was isolated from totalRNA by the use of the Poly AT tract System (Promega, USA). The isolatedmRNA was used in cDNA production.

For cDNA production, oligonucleotides were produced to the complimentarystrand of the hexon gene and the penton base gene, both being MLPtranscripts. A further ollgonucleotide was produced which covered theproposed cap site of the major late transcript, 24 bases downstream ofthe TATA box. This oligonucleotide was used in conjunction with thatused in cDNA production in Taq polymerase chain reaction. The resultingDNA produced from positive clones was digested with appropriaterestriction enzymes to determine the size of the inserted fragment. DNAsequencing of these inserted fragments was performed using amodification of the chain termination technique (Sanger, F., Nicklen, Sand Gulson, A. R., 1977, DNA sequencing with chain terminatinginhibitors. PNAS USA 74: 5463-5467) so as to allow Taq DNA polymeraseextension (Promega, USA).

To confirm the leader sequence cap site, fresh cDNA was prepared andthis time a tail of dGTP residues added to it. Briefly, cDNA wasincubated with 1 mM dGTP and approximately 15 units of terminaldeoxynucleotidyl transferase (Promega) in 2 mM CaCl2 buffer at 37° C.for 60 minutes. The reaction was stopped by heating to 70° C. for 10minutes. The DNA was then ethanol precipitated and resuspended in avolume suitable for use in polymerase chain reaction (PCR). PCR wasperformed as previously described using a poly (dC) oligonucleotide witha XbaI site at the 5′ end. Resulting fragments were blunt ended with T4DNA polymerase at 37° C. for 30 minutes in the presence of excessnucleotides and cloned into the SmaI site of the pUC18 vector. DNApreparation and sequencing were performed, as described previously, onclones shown to be positive by hybridisation.

FIG. 3 illustrates the separate sequences of the major late promoter,upstream enhancer sequence and splice leaders 1, 2 and 3 as determinedfrom cDNA studies. FIG. 2 illustrates the DNA sequence of the completepromoter cassette with the components joined together.

Characterisation of Non-essential Regions of Viral Genome

The right end was identified by cloning and complete sequencing of thePAV serotype 3 ApaI fragment J of approximately 1.8 Kbp. The invertedterminal repeat (ITR) has been determined by comparison of the RHEsequence with that of the left hand end. The ITR is 144 bases long andrepresents the starting point into which potential insertions can bemade. FIG. 4 shows the sequence of the terminal 720 bases. Restrictionendonuclease sites of interest for insertion of foreign DNA areindicated in the terminal sequence. A putative TATA site for the E4promoter is identified, this being the left most end for the possiblesite of insertion. Initial insertions will be made into the SmaI orEcoRI sites.

The E3 region of the genome, this also being a non-essential area, hasbeen located and cloned. The promoter region of E3 has been identifiedand the overlapping L4 area sequenced (FIG. 5). The region of the E3after the polyadenylation signal of the L4 is also a possible site forinsertion and can also be used for deletion to create more room forlarger cassette insertions.

Construction of PAV Vector

FIG. 6 illustrates a preferred method of construction of a PAV vector.The right hand end ApaI fragment J of PAV serotype 3 is cloned and aunique SmaI restriction endonuclease site 230 bp from the invertedrepeats was used as an insertion site.

The major late promoter expression cassette containing the E2 (gp55)gene of classical swine fever virus (hog cholera virus) was cloned intothe SmaI site of the RHE fragment.

A preferred method of homologous recombination was cutting genomic PAV 3DNA with HpaI, a unique site in the genome, and transfecting this DNAwith ApaI cut expression cassette plasmid containing gp55.

The DNA mix was transfected into preferably primary pig kidney cells bystandard calcium chloride precipitation techniques.

The preferred method of transfection generates recombinant virus throughhomologous recombination between genomic PAV 3 and plasmid (FIG. 6).

DETAILED DESCRIPTION OF THE INVENTION

Construction of PAV Vector

The following examples show the construction of representativerecombinant porcine adenoviruses of this invention. The recombinantviruses were propagated and titred on primary porcine kidney cells.

1 Construction of PAV-gp55

An expression cassette consisting of the porcine adenovirus major latepromoter, the classical swine fever virus (CSFV) gene (gp55) and SV 40polyA was inserted into the SmaI site of the right hand end (MU 97-99.5)of porcine adenovirus serotype 3 and used to generate in porcine primarykidney cells a recombinant PAV 3. The size of the expression cassettewas 238 kilobase pairs. No deletion of the genomic PAV 3 was made.Mammalian adenoviruses with intermediate genomes (˜36 kb) have beenshown to accommodate up to 105% of the wild-type genomic length, andgenomes larger than this size are either unpackageable or extremelyunstable, frequently undergoing DNA rearrangements (Betts, Prevec andGraham, Journal of Virology 67, 5911-5921 (1993), Packaging capacity andstability of human adenovirus type 5 vectors: Parks and Graham, Journalof Virology, 71, 3293-3298, (1997), A helper dependent system foradenovirus vector production helps define a lower limit for efficientDNA packaging). In this invention, PAV genomic length was 34.8 kb, intowhich was inserted without any other deletion an expression cassette of2.38 kb. The resulting genomic DNA length of the recombinant porcineadenovirus of this invention was 106.8%, and therefore exceeded theputative maximum limit for construction of a stable recombinant Therecombinant virus was plaque purified three times and passaged stably inprimary pig kidney cells. The recombinant was shown to contain gp55 bySouthern blot hybridisation. Expression of gp55 was demonstrated byinfecting primary PK cell line grown on glass cover slips with therecombinant porcine adenovirus. After 24 hours, immunoflouresencentstaining (IF) showed infected cells expressing gp55.

2 Construction of recombinant PAV-G-CSF

An expression cassette comprising of the porcine adenovirus moor latepromoter, the gene encoding porcine granulocyte-colony stimulatingfactor (G-CSF) and SV40 polyA was inserted into the SmaI site of theright hand end (MU 97-99.5) of porcine adenovirus serotype 3 and used togenerate in porcine primary kidney cells a recombinant PAV 3. The sizeof the expression cassette was 1.28 kilobase pairs. No deletion of thegenomic PAV 3 was made. The recombinant virus was plaque purified twotimes and passaged stably in primary pig kidney cells. The recombinantwas shown to contain G-CSF by Southern blot hybridisation and polymerasechain reaction (PCR). Expression of G-CSF was demonstrated by infectingprimary kidney cells with the recombinant PAV-G-CSF. Tissue culturesupernatants from the infected primary kidney cells were thenelectrophoresed in SDS-PAGE gels and transferred to filters. Infectedcells expressing G-CSF were detected in a Western blot using a rabbitpolyclonal antiserum against porcine G-CSF expressed by purifiedrecombinant E coli.

3 Construction of recombinant PAV-gp55T/GM-CSF

An expression cassette consisting of the porcine adenovirus moor latepromoter, a truncated form of the classical swine fever virus gene gp55fused in frame to the gene encoding either the full length or the matureform of porcine granulocyte/macrophage-colony stimulating factor(GM-CSF) and SV40 polyA was inserted into the SmaI site of the righthand end (MU 97-99.5) of porcine adenovirus serotype 3 and used togenerate in porcine primary kidney cells a recombinant PAV 3. The sizeof the expression cassette was 2.1 kilobase pairs. No deletion of thegenomic PAV 3 was made. The recombinant virus was plaque purified twotimes and shown to contain gp55 and GM-CSF by PCR.

4 Construction of Recombinant PAV-gp55/E3

The insertion vector pJJ408 containing the right hand end ApaI fragmentJ of the PAV serotype 3 genome (approximately 1.8 kbp), was enlarged tocontain the complete BglII B fragment comprising 7.2 kbp of the PAV3right hand end (FIGS. 15 a and b). This fragment contains both the righthand end insertion site described previously and the E3 region. Theright hand end insertion site was engineered to contain the PAV3 MLP/TPLsequences followed by a multiple cloning site and the SV40 poly Asequence.

An E3 insertion site was constructed by excising a 622 bp SnaBI/BsrGIfragment within the E3 region of the PAV serotype 3. TheMLP/TPL-gp55-Poly A expression cassette was inserted into theSnaBI/BsrGI site (FIGS. 15 b and c). This plasmid was used intransfections to produce a recombinant PAV3 containing theMLP/TPL-gp55-poly A cassette inserted in the partially deleted E3 region(FIG. 15 c).

Wild type PAV3 DNA was digested with SnaBI restriction enzyme yieldingtwo fragments of 28.712 kbp and 5.382 kbp. The large left hand fragmentwhich includes the overlap region of the right hand end and the lefthand end of the PAV3 genome was gel purified. This fragment wastransfected into primary PK cells along with KpnI restricted E3/rheinsertion vector DNA in 3 cm petri dishes to allow homologousrecombination to occur between the PAV3 and insertion vector DNA. Usingthis method, only recombinant virus are recovered.

Cells were maintained for 5 days at 37° C. and then frozen and thawedtwice. Lysate was passaged into fresh primary PK cells and observed forthe development of plaques. The recombinant virus was plaqued purifiedand shown to contain gp55 by PCR.

Vaccination Strategy

1. Vaccination with PAV-gp55

In this experiment 5-6 week old piglets were used to representimmunocompetent pigs. A group of the piglets (#2, 6 and 7) werevaccinated with recombinant PAV-gp55 administered subcutaneously at adose of 1×10⁷ pfu per piglet A control group of piglets (#3, 8, 11, 12,13 and 14) were unvaccinated. No clinical signs were observed (no risein temperature) in the vaccinated group of piglets (Table 1).Temperatures post Vaccination with rPAV:: gp55 CSFV (° C.) Pig No. Day 01 2 3 6 9 10 13 2 39.7 39.2 39.4 39.8 39.6 39.8 39.6 39.2 3 39.5 39.239.4 39.0 38.8 39.3 39.0 39.7 (control) 6 39.7 39.1 39.1 39.0 39.1 39.839.1 39.8 7 39.4 39.8 39.8 39.4 39.9 38.9 39.6 39.7 8 39.6 39.5 39.439.0 40.5 39.4 39.1 39.7 (control)

Five weeks after vaccination with the recombinant PAV-gp55 both groupsof pigs were challenged with a lethal dose (1×10^(3.5) TCID₅₀) ofvirulent Hog Cholera virus (Classical swine fever virus) appliedsubcutaneously.

The temperatures of the pigs were monitored and the results tabulated inTable 2 and graphically represented in FIG. 7. TABLE 2 Temperatures postchallenge with CSFV (° C.) Day Pig No. −1 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 2 39.6 39.9 40.1 40.3 40.1 39.2 39.7 39.5 39.5 39.7 39.4 39.139.5 39.1 40.0 39.4 38.9 3 39.6 40.4 39.6 40.0 40.7 40.7 41.9 40.7 40.942.0+ 6 39.5 39.5 40.0 40.0 39.6 40.5 39.9 39.2 39.2 38.8 39.3 38.8 38.939.6 39.3 39.1 38.9 7 39.8 39.9 40.4 40.6 40.3 39.7 39.7 39.7 39.5 39.339.1 39.3 39.6 40.6 39.7 39.8 39.7 8 39.9 40.6 40.5 40.3 40.0 41.4 39.841.0 40.6 39.0+ 11 39.6 39.9 40.0 40.3 40.7 40.5 40.0 41.8 41.5 41.3+ 1239.8 39.9 40.9 41.0 41.2 40.6 40.1 41.0 41.7 40.3+ 13 39.7 40.0 41.241.5 41.6 41.0 39.7+ 14 39.3 40.0 39.6 39.8 40.3 40.7 41.2 40.8 40.241.7+

The results show that by day 5 the control group had elevatedtemperatures (greater than 40.5° C.) and showed clinical signs ofdisease. The vaccinated group showed no clinical signs of disease. Pigsfrom the control group were dead or euthanased by day 9. The vaccinatedgroup were euthanased at day 16. At post mortem all control pigs showedsevere clinical disease, the vaccinated pigs showed no clinical signs ofdisease.

The results indicate that the pigs vaccinated subcutaneously with therecombinant PAV-gp55 survived challenge with classical swine fever virusat a lethal dose.

Sera were collected from both groups of pigs and tested for the presenceof antibodies to PAV by ELISA. These tests showed the presence ofpre-existing antibodies to PAV before vaccination. The level of theseantibodies increased following vaccination with the recombinant PAV-gp55to peak between days 28 and 36 post vaccination. These results aretabulated in FIG. 8.

Sera were collected from the vaccinated group of pigs pre and postchallenge with CSFV and tested in the presence of neutralisingantibodies to CSFV. Sera were tested at days 0 and 28 after vaccinationwith recombinant PAV-gp55 (pre challenge) and then again at day 16 postchallenge (day 52 after vaccination). The results in FIG. 9 show noneutralising antibodies detected at day 0, low levels of neutralisingantibodies at day 28 and high levels at day 52.

These results show that the recombinant PAV-gp55 can protect pigs fromlethal challenge with classical swine fever virus in the presence ofpre-existing antibodies to PAV.

2. Vaccination with PAV-G-CSF

In this experiment 5-6 week old piglets were used to representimmunocompetent pigs. A group of pigs (n=4) were vaccinated withrecombinant PAV-G-CSF administered subcutaneously at a dose of 1×10⁷ pfuper piglet A second group (n=4) were vaccinated with PAV wild type (wt)administered subcutaneously at a dose of 1×10⁷ pfu per piglet A controlgroup (n=4) were unvaccinated. Pigs were bled at 8 hour intervals for aperiod of 104 hours post vaccination. Complete blood counts weredetermined and the mean white blood cell (WBC) counts for each groupmonitored. These results are graphically represented in FIG. 10 and thepercentage change in mean WBC counts graphically represented in FIG. 11.

Pigs vaccinated with either PAV wt or PAV-G-CSF showed clinical signs ofdisease with mild diahorrea 24-72 hours post vaccination. Both groups ofpigs were completely recovered by 80-96 hours post-vaccination. Controlpigs showed no clinical signs of disease.

Complete blood screening results show that the mean WBC counts forcontrol pigs increased over the duration of the experiment.

PAV wt vaccinated pigs also show an increase in WBC counts, with adepression in WBC counts between 48-80 hours post-vaccination andrecovery from 80-96 hours onwards.

Pigs vaccinated with the recombinant PAV-G-CSF show a significantdepression in WBC counts over the duration of the experiment Astatistical analysis of these results is tabulated in Table 3. Theanalysis shows that differences between the mean WBC counts (controlsand PAV-G-CSF; PAV wt and PAV-G-CSF) were significant indicating thatthe recombinant PAV-G-CSF altered the proportions of cells involved withimmunity. TABLE 3 Results of t-tests between mean WBC counts of groupsof pigs vaccinated with either PAV wild type (wt), PAV recombinantexpressing G-CSF (PAV-G-CSF) or unvaccinated controls. Pre vacc 0 hr8-24 hr^(d) 32-48 hr 56-72 hr 80-104 hr Control p > 0.2^(b) P > 0.2 P >0.2 P > 0.2 P < 0.005 vs PAV-G- CSF^(a) Control p > 0.1 p > 0.01^(c) p >0.02 p > 0.2 P < 0.05 vs PAV wt PAV-G-CSF p > 0.2 p > 0.05 p > 0.05 P <0.05 P < 0.001 Vs PAV wt^(a)null hypothesis; them is no difference between the mean WBC counts.^(b)p > 0.05, insufficient to reject the null hypotheses at the 95%confidence level, conclude that there is no difference between meanleucocyte levels.^(c)p < 0.05, null hypothesis rejected at 95% confidence level, concludethat there is a difference between the mean leucocyte levels.^(d)4 pigs in each group were bled at 8 hour intervals.

Differential WBC counts were also determined and monitored for eachgroup. The percentage change in mean monocyte cell populationsgraphically represented in FIG. 12 and the percentage change in meanlymphocyte cell populations graphically represented in FIG. 13. FIG. 12shows that monocyte cell populations increased rapidly in pigs followingvaccination with PAV wt, but were suppressed by vaccination with therecombinant PAV-G-CSF. This effect was due to the expression of G-CSF bythe recombinant. A statistical analysis of these results is tabulated inTable 4. The analysis shows that there was a significant differencebetween the PAV wt and PAV-G-CSF from 32 to 96 hours post vaccination.FIG. 13 shows that there were shifts in lymphocyte cell populationnumbers following vaccination with the recombinant PAV-G-CSF.Unvaccinated controls show stable lymphocyte cell numbers over theduration of the experiment, whereas pigs vaccinated with PAV wt show asignificant increase in lymphocyte cell population as a response toinfection.

Pigs vaccinated with the recombinant PAV-G-CSF show a decline inlymphocyte cell population. A statistical analysis of these results istabulated in Table 5. The analysis shows that there was a significantdifference between PAV wt and the recombinant PAV-G-CSF between 8 and 96hours post vaccination. The different responses in lymphocyte cellproliferation following vaccination with recombinant PAV-G-CSF and PAVwt were due to the expression of G-CSF by the recombinant These resultsshow that vaccination with recombinant PAV-G-CSF produces a shift insub-populations of cells involved in immunity. TABLE 4 Results oft-tests between mean monocyte cell populations following vaccination ofpigs with either recombinant PAV-G-CSF, wild type PAV (PAV wt) orunvaccinated controls. Pre vacc 8-24 hr^(d) 32-48 hr 56-72 hr 80-96 hr104 hr Control p > 0.1^(b) P > 0.2 P > 0.2 P > 0.2 P > 0.2 p > 0.2 VsPAV-G-CSF^(a) Control p > 0.2 P < 0.002^(c) p > 0.2 P < 0.001^(c) P >0.2 p > 0.2 Vs PAV wt PAV wt p > 0.2 P < 0.001 p > 0.2 P > 0.2 P > 0.2p > 0.05 Vs PAV-G-CSF^(a)null hypothesis; there is no difference between the mean monocytecell counts.^(b)p > 0.1, insufficient to reject the null hypothesis at the 90%confidence level, conclude that there is no difference between meanmonocyte cell levels.^(c)p < 0.05, null hypothesis rejected at 95% confidence level, concludethat there is a difference between the mean monocyte cell levels^(d)4 pigs in each group were bled at 8 hour intervals

TABLE 5 Results of t-tests between mean lymphocyte cell populationsfollowing vaccination of pigs with either recombinant PAV-G-CSF, wildtype PAV (PAV wt) or unvaccinated controls. Pre vacc 8-24 hr^(d) 32-48hr 56-72 hr 80-96 hr 104 hr Control p > 0.2 P > 0.05^(b) P > 0.2 P > 0.2P > 0.2 p > 0.2 Vs PAV-G-CSF^(a) Control p > 0.2 P > 0.2 P < 0.01^(c) P< 0.001^(c) P < 0.001^(c) p > 0.2 Vs PAV wt PAV wt p > 0.2 P < 0.05^(c)P < 0.002^(c) P < 0.005^(c) P < 0.001^(c) p > 0.05 Vs PAV-G-CSF^(a)null hypothesis; there is no difference between the mean lymphocytecell counts.^(b)p > 0.05, insufficient to reject the null hypothesis at the 95%confidence level, conclude that there is no difference between meanlymphocyte cell levels.^(c)p < 0.05, null hypothesis rejected at 95% confidence level, concludethat there is a difference between the mean lymphocytecell levels.^(d)4 pigs in each group were bled at 8 hour intervals

FIG. 14 graphically represents changes in the proliferation of T cellsof each group following stimulation with Concanavalin A (Con A). Theseresults confirm that there was a significant proliferation of T-cellsfollowing vaccination with PAV wt at day 2 post vaccination, whereasvaccination with the recombinant PAV-G-CSF resulted in a suppression ofT-cell proliferation by day 3.

The results of vaccination with a recombinant PAV expressing porcineG-CSF shows that G-CSF has a significant effect on the cells involvedwith immune responses.

It will be appreciated that whilst this document establishes the metesand bounds of this invention, all embodiments falling within its scopefor example with regard to heterologous genes, insertion sites, types ofpromoter and senotype have not necessarily been specifically exemplifiedalthough it is intended that they should fall within the scope ofprotection afforded this invention.

FIG. 2

Total sequence of the PAV Major Late Promoter cassette including theadded nucleotides 5′ (upstream) of the USF. Nucleotide base count: 76 A143 C 187 G 96 T Total 502 bp 1 GGTGCCGCGG TCGTCGGCGT AGAGGATGAGGGCCCAGTCG GAGATGAAGG CACGCGCCCA 61 GGCGAGGACG AAGCTGGCGA CCTGCGAGGGGTAGCGGTCG TTGGGCACTA ATGGCGAGGC 121 CTGCTCGAGC GTGTGGAGAC AGAGGTCCTCGTCGTCCGCG TCCAGGAAGT GGATTGGTCG 181 CCAGTGGTAG TCCACGTGAC CGGCTTGCGGGTCGGGGGGT ATAAAAGGCG CGGGCCGGGG 241 TGCGTGGCCG TCAGTTGCTT CGCAGGCCTCGTCACCGGAG TCCGCGTCTC CGGCGTCTCG 301 CGCTGCGGCT GCATCTGTGG TCCCGGAGTCTTCAGGTCCT TGTTGAGGAG GTACTCCTGA 361 TCGCTGTCCC AGTACTTGGC GTGTGGGAAGCCGTCCTGAT CGCGATCCTC CTGCTGTTGC 421 AGCGCTTCGG CAAACACGCG CACCTGCTCTTCGGACCCGG CGAAGCGTTC GACGAAGGCG 481 TCTAGCCAGC AACAGTCGCA AGThe Upstream Stimulatory Factor (USF) and TATA motiff are in bold. Thecomplete leader sequence is italised with the cap site and splice sitesbetween the individual leaders indicated by double underlining or singleunderlining respectively.FIG. 3Individual sequences of the Promoter cassette components:

I. The 5′ (upstream) sequence included in the long cassette. 1GGTGCCGCGG TCGTCGGCGT AGAGGATGAG GGCCCAGTCG GAGATGAAGG CACGCGCCCA 61GGCGAGGACG AAGCTGGCGA CCTGCGAGGG GTAGCGGTCG TTGGGCACTA ATGGCGAGGC 121CTGCTCGAGC GTGTGGAGAC AGAGGTCCTC GTCGTCCGCG TCCAGGAAGT GGATTGGTCG 181CCAGTGGTAG

II. Sequence including the USF, TATA motiff and sequence to the capsite. 1 CCACGTGACC GGCTTGCGGG TCGGGGGGTA TAAAAGGCGC GGGCCGGGGTGCGTGGCCGT 61 C

III. First leader sequence. 1 AGTTGCTTCG CAGGCCTCGT CACCGGAGTCCGCGTCTCCG GCGTCTCGCG CTGCGGCTGC 61 ATCTGTGGTC CCGGAGTCTT CAG

IV. Second leader sequence. 1 GTCCTTGTTG AGGAGGTACT CCTGATCGCTGTCCCAGTAC TTGGCGTGTG GGAAGCCGTC 61 CTGATCG

V. Third leader sequence. 1 CGATCCTCCT GCTGTTGCAG CGCTTCGGCA AACACGCGCACCTGCTCTTC GGACCCGGCG 61 AAGCGTTCGA CGAAGGCGTC TAGCCAGCAA CAGTCGCAAGFIG. 4Sequence of the right hand end of the PAV genome this area being aproposed site for insertion of expression cassettes.

Nucleotide base count 183 A 255 C 306 G 204 T Total 948 bases 1CATCATCAAT AATATACCGC ACACTTTTAT TGCCCCTTTT GTGGCGTGGT GATTGGCGGA 61GAGGGTTGGG GGCGGCGGGC GGTGATTGGT GGAGAGGGGT GTGACGTAGC GTGGGAACGT 121GACGTCGCGT GGGAAAATAA CGTGGCGTGG GAACGGTCAA AGTCCGAGGG GCGGGGTCAA 181AGTCCGCAGT CGCGGGGCGG AGCCGGCTGG CGGGAATTCC CGGGACTTTC TGGGCGGGTA                                 EcoRI      SmaI 241 ATCGTTAACGCGGAGGCGGG GGAATTCCGA TCGGACGATG TGGTACTGAT TAACCGACCGHpaI                  EcoRI 301 CAGGCGTGTC CACATCCGCT GTGGGTATATCACCGGCGCT CGCGGTGTTC GCTCACACTC 361 GTCTCGGCGC TGTCACAGAG AGAGACACTGAGAGCGAGAC GAGGAGAAAC CGAAAGCGGG 421 GCAGGAGGAG TCACCGGGCC ATCTTCCCATCAGAGCCCTC TCATGGCCCA CGACCGACTG 481 CTGCTGGCCG CGGTGGCTGA CTGTTGCTCGCCGTGCTCTA TCTGTACTTC GCCTACCTCG 541 CGTGGCAGGA TCGGGACACT CTTCACACTCAGGAGGCCGC CTCTCCTCGC TTCTTCATCG 601 GGTCCAACCA CCAGCCCTGG TGCCCGGATTTTGATTGGCA GGAGCAGGAC GAGCACACTC 661 ACTAGACGTT TAGAAAAAAG ACACACATTGGAACTCATAT ATGTCTGCGG GACCGCATCA 721 GCAGCCCGGT CTGCTGTTGG CTGCGGGTGAGAGGCCTCCG GTAATTCATC AGAACCGCAT                                  StuI781 TCATCTGCGC CACGTCCCGA CATATGGTGC TGACGTCAGA ACAGCCCAGC GTGATCCTTT                                 SacIII 841 TAATGTGCTA GTCTACGTGCCCACTGGGTT TGCTGTGTTT GTGCCGACTG AGCGAGATTT 901 TCAGAGGAGG GATCTGGTCCGTTTCCAGAC CTGCTGCTTC CGGCATCAThe Inverted Terminal Repeat (ITR) is shown in bold. Enzyme sites ofinterest are underlined with the enzyme name below. Putative TATA for E4region is also shown.

1-42. (canceled)
 43. A method of vaccination of pigs against diseaseincluding administering to said pigs a first recombinant porcineadenovirus vector stably incorporating, and expressing a heterologousnucleotide sequence encoding at least one antigenic determinant of saiddisease against which vaccination is desired, wherein said heterologousnucleotide sequence is inserted into a non-essential region of a siteselected from the group consisting of the E3 region and map units97-99.5 of PAV3.
 44. A method as claimed in claim 39 includingadministering to said pig a second porcine adenovirus vector includingat least one heterologous nucleotide sequence which differs from aheterologous nucleotide sequence incorporated in said first recombinantporcine adenovirus vector.
 45. A method as claimed in claim 40 whereinsaid second porcine adenovirus vector incorporates, and is expressing ofat least one heterologous nucleotide sequence encoding animmuno-potentiating molecule.